Special application notecyBi®-FeliX

In cooperation with scienova1

AbstrAct

The CyBi®FeliX automated liquid handling system with Head R 96/250 µl was used in conjunction with the Scienova MD100 GridKit48 for complete dialysis process of enzyme reactivation through urea removal and protein recovery. The procedure was shown to significantly increase sample throughput and processing accuracy.

IntroductIon

The removal of denaturing agents can be time-consuming and difficult. Therefore the Scienova GmbH offers a simple, cost-efficient and fast dialysis system in the popular 96-well microplate format. Through their patented design and low-binding regenerated cellulose membranes those “Xpress Micro Dialyzers” (MD) are easy to handle and have excellent sample recoveries. They can be used for an extensive variety of applications like:• Removalofdenaturatingagentsfromsamples• Sampledialysesofproteins,oligonucleotides,RNAorDNA• Bufferexchange,rebuffering• Removalofdyes• Desalting• Sampleconcentration

To simplify and improve the dialysis process, Xpress Micro Dialyzers is combined in a kit which enables the simultaneous handling of up to 48 samples without human intervention.

Automation of the scienova Micro dialyzer Md100 GridKit48 on cybi®-FeliX

J. Groß, s. Kreusch Phd; scienova GmbHK. undisz Phd; Analytik Jena AG

Figure 1: One cartridge of micro dialyzers MD100 with 8 micro dialyzer units

Special application notecyBi®-FeliX

In cooperation with scienova2

Trypsinisreversiblyinhibitedinthepresenceofhighureaconcentrations.Toregaintrypticactivity,byparanitroaniline(PNA)release,urea removal is needed, which is achieved through dialysis.

DL– benzoyl– Argp– nitroanilineTrypsinp – nitroaniline+benzoyl– Arg

Toshowthatthereisnosamplelossinconsequenceofproteinbindingwiththemembrane,differentbovineserumalbumin(BSA)solutions were dialyzed for two hours and the recollected samples were analyzed.

cyBi®-FeliXThe CyBi®-FeliX is a multi-channel/single-channel pipettor for automated liquid handling. Depending on the specific application needs different pipetting heads and liquid handling adapters are available. The CyBi®-FeliX with the Head R 96/250 µl enables a simultaneous handling of up to 96 samples on 12 deck positions.

Scienova MD100 GridKit48The Scienova GridKit48 consists of a 48-deep well plate, a grid and six cartridges à eight Micro Dialyzers (48 samples) with the chosen cutoff (3.5, 6-8, 12-14 kDa).By the help of the grid, all Micro Dialyzers have a well-defined positionandagoodgrip.Additionallythefreepositionsinthegrid can be used for buffer handling while dialysis. For the following experiments MD 100 cartridges with a cutoff from 6-8 kDa and a maximal sample volume of 100 µl were used.

MAterIAls And MetHods

Figure 2: CyBi®-FeliX with Head R 96/250 µl in combination with Scienova MD 100 GridKit48

Figure 3: Scienova MD100 GritKit48

AIM

Special application notecyBi®-FeliX

In cooperation with scienova3

Micro Dialyzers were placed in 4.4 ml dialysis buffer into the grid and filled with 100 µl trypsin sample by CyBi®-FeliX. Forty samples (n=40)withureaandeightsamples(n=8)withoutureaasreferenceweredialyzed60minatroomtemperature(22°C).Afterincubation time the samples were transferred to a 96-well microplate for measurement.

MicroDialyzerswereplacedin4.4mlA.deion.intothegridandfilledwith100µlBSAsamplebyCyBi®-FeliX. For each concentration sixsamples(n=6)andtworeferences(n=2)weredialyzedfor120minatroomtemperature(22°C).AfterIncubationtimethesampleswere transferred to a 96-well microplate for measurement.

experiment 1: enzyme reactivation through urea removal

Dialysis samples 100 µl trypsin samples:1. 0.5 mg/ml trypsin in 8 M urea, 20 mM CaCl22. 0.5 mg/ml trypsin in 20 mM CaCl2 as reference

Dialysis buffer 4.4 ml of dialysis buffer(35 mM Tris pH 7.8, 20 mM CaCl2)

Measurement solution 200 µl(4.7mMDL-Benzoyl-Argp-nitroaniline(DLBAPNA)in10%DMSO+trypsin0.05mg/mlin35mMTrispH7.8, 20 mM CaCl2)

Determination method Photometer BioTek ELx800, 405 nm (measurement wavelength) and 620 nm (reference wavelength)

PNA-releaserate(rr)indicatingtrypticactivityisevaluatedas:rr=absorbance 405 nm – absorbance 620 nm

∆t in min“

Urea determination WescorVAPRO5520Osmometer

experiment 2: BSa recovery

Dialysis samples 1.100µlofBSAsolutionin8MUrea(n=6)2.100µlofBSAsolutioninA.deion.(n=2)asreferenceforeachconcentration:10,20,40,60,80and100µg/ml

Dialysis buffer 4.4mlofA.deion.

Determination methods

Protein determination according to Bradford, Tecan Sunrise photometer: λ (590/450 nm)Ureadetermination:WescorVAPRO5520Osmometer

Special application notecyBi®-FeliX

In cooperation with scienova4

results enzyMe reActIvAtIon

ThefollowingtableshowsthePNA-releaserate(rr)dependingontheureaconcentration.

Afteronehourofdialysistheureaconcentrationdroppedfrom8Mto0.30M.

Theresultsshowthataregainofabout75%trypticactivitycouldbeachieved after one hour. In total all 48 samples have a low standard de-viation which indicates a good constancy and reproducibility. The combination of Scienova MD100 GridKit48 and CyBi®-FeliX enables a high sample throughput without losing quality, useable for all applications of Scienova Micro Dialyzers.

sample non-dialyzed sample 60 min dialyzed reference dialyzed reference non-dialyzed

MeanPNAreleaserate(1/min)

0.0045 0.0099 0.0130 0.0131

Standard deviation 0.0001 0.0006 0.0014 0.0008

Urea concentration (M) 8 0.30 0 0

Figure 4: Regain of tryptic activity

Figure 5: Urea Removal

sample non-dialyzed

samples 60 min dialyzed

reference 60 min dialyzed

reference non-dialyzed

0.016

0.014

0.012

0.01

0.0008

0.0006

0.0004

0.0002

0

PNA

rr[1

/min

]

Special application notecyBi®-FeliX

In cooperation with scienova5

results bsA recovery

The following figures contain all recovery values compared with the references.

Aftertwohoursofdialysistheureaconcentrationdroppedfrom8Mto0.23M.Therecoveryvaluesdisplayarangeof87to90%independent from the protein concentration.

sample non-dialyzed sample 60 min dialyzed reference dialyzed reference non-dialyzed

MeanPNAreleaserate(1/min)

0.0045 0.0099 0.0130 0.0131

Standard deviation 0.0001 0.0006 0.0014 0.0008

Urea concentration (M) 8 0.30 0 0

Figure 6:ExtinctionofdifferentBSAsamplesat590nm Figure 7:BSArecoveryin%

dIscussIon

AsaresultofrepeatedimplementationtheBSArecoveryratesweresignificantlysmaller(upto5%).Therearenosignificantdifferencesbetween automated and manual process. One reason for a difference is the high mobility of urea in solutions and the short diffusion distances in Scienova Xpress Micro Dialyzer. There is only a minor osmotic effect, resulting in a slightly higher volume recovery of about 110µl.Anadvantageofautomationisthesmallrangeofvariationwhichalsoexistsforvolumesdownto25µl.Themanualfillingandemptying of 48 micro dialyzers might take up to 20 min, whereas the CyBi®-FeliXmanagesitinlessthanoneminute.Alsomanualpipettingerrorswillbeeliminated.ThisBSAassayisanexampletoshowthestabilityofproteinsinsidetheMicroDialyzer.Itisrecommendedto make a pretest, because other proteins maybe behave differently. The enzyme reactivation shows, that an immediately automated removal by dialysis of denaturing agents is possible. Based on the results, the Scienova MD100 GridKit48 is one of the first disposables for a complete automation of dialysis process. The accurate height adjustment in tenth of millimeter and the simultaneous handling by CyBi®-FeliX of up to 48 samples enables a high throughput of samples to dialyze, efficiency can be further increased by automated mixing or changing the buffer while dialysis is running.

BSAin8MUreadialyzedBSAinAquadeion.dialyzedBSAinAquadeion.non-dialyzed

1.0000.9000.8000.7000.6000.5000.4000.3000.2000.1000.000

105.00

100.00

95.00

90.00

85.00

80.00

75.00

70.00

65.00

60.00

Exti

nktio

n 59

0 nm

BSA

Rec

over

y[%

]10 µg/ml 20 µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100 µg/ml

10 µg/ml 20 µg/ml 40 µg/ml 60 µg/ml 80 µg/ml 100 µg/ml

BSAin8MUreadialyzed BSAinAquadeion.dialyzed

Special application notecyBi®-FeliX

Analytik Jena AGKonrad-Zuse-Str. 1 Phone: +49 (0) 36 41/77 70 info.cybio@analytik-jena.com07745 Jena/Germany Fax: +49 (0) 36 41/77 92 79 www.cybio-ag.com M

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reference to the source.

This documentation describes the state at the time of publishing. It needs not necessarily agree with future versions. Subject to change! Technical modifications, misprint and errors excepted! Expression and further use permitted with indication of source. © 2015 Analytik Jena AG

In cooperation with scienova